Abstract
Secretory granules (SGs) in mast cells contain various molecules that elicit allergy symptoms and are generally considered therapeutic targets. However, the biogenesis, maintenance, regulation, and recycling of these granules remain controversial, mainly due to the lack of suitable live-cell imaging methods. In this study, we applied negative contrast imaging with soluble green fluorescent protein (GFP) expressed in the cytoplasm as a method to validate structural information of mast cell SGs. We evaluated the accuracy of the method in detail, and we demonstrated that it can be used for quantitative analysis. Using this technique, secretory granules, the nucleus, mitochondria, and the cell body were visualized in individual RBL-2H3 mast cells without any influence. When combined with conventional multicolor fluorescence imaging, visualization of SG-associated proteins and SG–SG fusion was achieved. Moreover, 3D images were constructed based on this method, and detailed information on the number, size, and shape of individual SGs was obtained. We found that cell volume was correlated with SG number. In summary, the technique provides valuable and unique data, and will therefore advance SG research.
Highlights
IntroductionOrganelles including SGs are separated from the cytoplasm by a lipid bilayer
The generic SG markers neuropeptide Y (NPY) and CD63 mostly localized to areas of high negative contrast (Fig. 1b,c), indicating that these organelles are SGs
We applied negative contrast imaging (NCI) to live mast cells, and we were able to directly visualize all SGs with sufficient contrast to reconstruct 3D images. We anticipate that this method will facilitate molecular studies of SG biogenesis in mast cells
Summary
Organelles including SGs are separated from the cytoplasm by a lipid bilayer As these organelles exclude soluble fluorescent protein expressed in the cytoplasm, negatively stained organelles can be visualized by evanescent-field micrography[22], two-photon microscopy[23], and confocal microscopy[21,23,24]. We combined NCI with conventional fluorescence imaging to visualize interactions between SGs and associated proteins, and to observe SG–SG fusion between marked and unmarked granules. We reconstructed 3D images of individual mast cells, and found a correlation between cell volume and SG number, and between SG volume and SG number This method enables real-time observation of SG development, as well as the analysis of associated cell structures
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